Prevalence of class 1, 2 and 3 integrons among multidrug-resistant Pseudomonas aeruginosa in Yazd, Iran.

Background and Objectives
Antibiotic resistance in Pseudomonas aeruginosa is an increasing health problem. Integrons are associated with a variety of gene cassettes, which confer resistance to multiple classes of antibiotics. This study aimed at screening the presence of class 1, 2 and 3 integrons in P. aeruginosa in Yazd, Iran.


Materials and Methods
This study was carried out on P. aeruginosa strains from March 2016 to March 2017. Clinical specimens were initially identified by the standard biochemical methods and their resistance patterns to antibiotics were studied using the disc diffusion method. PCR was carried out for the detection of class 1, 2 and 3 integrons using intI1, intI2 and intI3 gene primers, respectively.


Results
Antimicrobial susceptibility test showed that 75% of isolates were detected as multi-drug resistant (MDR), and lowest resistance was observed in ciprofloxacin (48.6%) and most resistance was in gentamicin (63.2%). Moreover, PCR results showed that 22 (15.3%) and 119 (82.6%) of P. aeruginosa isolates carried intI2 and intI1 genes, but intI3 gene was not found.


Conclusion
Since it is customary to observe Class I integrons in P. aeruginosa isolated from clinical samples, they are often responsible for antibiotic resistance gene transfer, which calls for evaluation of integrons as contributing factors in antibiotic resistance.


INTRODUCTION
Pseudomonas aeruginosa are the leading Gram-negative pathogen bacteria associated with nosocomial infections (1). P. aeruginosa is responsible for 10-15% of the nosocomial infections worldwide (2). Antimicrobial resistance is a worldwide emerging problem (3), and the widespread use of antibiotics is probably the main reason for the increase in multidrug resistance (MDR) among P. aeruginosa strains (4). Besides, in many bacteria, exchangeable genetic elements such as plasmids, transposons and integrons are responsible for the dissemination of antibiotic resistance (5). Integrons are common systems of gene capture and expression that incorporate open reading frame and convert them into functional genes. Integrons are mainly composed of the promoter, attachment site (att I) and integrase gene (int I) (6). There has long been an association between three antibiotic resistant integron classes (including classes 1 to 3) and MDR phenotypes (7)(8), where classes are determined based on sequence differences in the respective intI gene (9). However, class 1 integrons have been found to be the most prevalent in clinical isolates, carrying single or multiple gene cassettes, which confer resistance to aminoglycosides, β-lactams, chloramphenicol, and macrolides (10)(11). Class 2 integrons are not as widespread among bacteria, even though class 2 integrons are associated with a mobile DNA element, the Tn7 transposon (12). Arakawa recognized a class 3 integron-mediating IMP-1 for the first time in an S. marcescens strain isolated in Japan in 1995, which is one of the few instances of carriage of that class of integrons reported so far (13). Class 2 and class 3 integrons contain the integrase genes (intI2 and intI3), whose products are 46% and 61% identical to class 1 integrase, respectively (5). In this respect, prevalence of integrons is variable in different parts of the world. For example, studies carried out in Malaysia, China, and Iran (14)(15)(16) showed respectively that 63%, 38% and 35.6% of isolated P. aeruginosa carry class 1 integron gene.
The aim of the present study was to find out the molecular relation and the existence of integrons with multidrug resistance pattern of P. aeruginosa strains isolated from clinical specimens in Yazd, Iran.

Specimen collection and identification.
This descriptive study was carried out on 144 clinical isolates of P. aeruginosa which were collected from Shahid Sadoughi Hospital, Yazd, from March 2016 to March 2017. A questionnaire was used for recording patient demographic data such as: name, age, sex, type of sample, and ward. Bacterial isolates were recovered from different clinical specimens such as blood, bronchial fluid, urine, cerebrospinal fluid, catheter, pleural fluid, ear swap, sputum, and wound and inoculated on sheep blood agar and EMB agar media before incubation at 37°C for 24 h. All isolates were identified as P. aeruginosa using standard biochemical tests like growth on Cetrimide agar medium (Liofilchem, Italy), growth at 42°C, oxidase test, gram stain, pigment production, and Oxidation/ Fermentation test.
PCR assay. Extraction of genomic DNA from P. aeruginosa isolates was performed by salting out method and were stored at -20°C until use (18). Specific primers were developed for each gene using Primer 3 (Table 1). The final optimized PCR reaction consisted of 0.5 μl MgCl 2 (100 mM), 0.5 μl dNTP (10 mM), 0.2 µl (1 unit) Taq DNA polymerase (Cinnagen, Iran), 1 µl of each primer (10 pmol) (Alpha DNA, Canada), 2.5 µl PCR buffer (10 X), and 0.5 μl of DNA template (100 μg/ml) in total volume of 25 μl with double distilled water. DNA amplification was performed in the thermo cycler (Quanta Biotech, England) using an initial denaturation step for 5 min at 94°C (one cycle), followed by 35 cycles of 1 min at 94°C, 1 min at 50°C and 1min at 72°C for intI1. The above condition was used for intI2 and intI3 the same as the one for intI1 but in annealing temperature at 47°C and 52°C, respectively. All of the reactions were finalized for 5 min at 72°C (one cycle). Amplicons were analyzed by electrophoresis on 1% agarose gels at 5V/Cm alongside with 50 bp DNA ladder. To confirm the amplicons, some isolates were sequenced and analyzed with bioinformatics software such as BLAST and ClustalW2 (20).

Statistical analysis.
The data was analyzed using the SPSS18. The Chi-square test was employed to calculate the P-value in terms of resistant numbers of integron-positive and integron-negative isolates (P<0.05).
Class 1 integron was detected in 119 (82.6%) isolates ( Fig. 1), of which 59.7% were multidrug-resistant and 22.9% were intermediate or sensitive to tested antibiotics. Class 2 integron was found in 22 (15.3%) P. aeruginosa isolates ( Fig. 1) of which 14.58% were multidrug-resistant, 0.7% were intermediate or sensitive, and 19 (13.19%) strains had both intI1 and intI2 genes. No intI3 gene was detect-ed in any of the isolates. A significant correlation was observed between the presence of integrons class 1 and resistance against gentamicin (Table 2), and also integrons class 2 and resistance against ceftazidime, amikacin and tobramycin (Table 3). Moreover, there was a significant correlation between burn unit and the presence of class 1 integrons.
Nucleotide sequence accession numbers. The nucleotide sequence data reported in this work was assigned to the NCBI/GenBank nucleotide sequence database under accession no. KF146819 and KF358999.

DISCUSSION
Currently, in the investigation of genetic bases of P. aeruginosa multi-resistance, an important aspect that has been considered is the integron and the associated gene cassettes (19). The role of these elements in the horizontal acquisition and expression of genes, and as a gene reservoir, has been associated with the emergence of antibiotic resistance among clinical isolates of bacteria (21). The presence of populations of MDR strains among clinical isolates is a cause of concern for physicians applying empirical treatment, especially in serious cases of P. aeruginosa infections.
The results of our study showed that 40 strains (27.7%) of P. aeruginosa isolated from our hospital were resistant to all commonly used antibiotics. Based on studies by Kohanteb (22) and Nikokar (23), it was found that 26.7% and 19.7% of the P. aeruginosa isolates were resistant to all anti-pseudomonas antibiotics. Our results are consistent with the findings of other studies. Furthermore, the results of this study showed that there were MDR P. aeruginosa strains disseminated through different clinical wards in our hospital, indicating lack of appropriate supervision on this issue at this hospital, thus the infection control measures should be applied to prevent the transmission of P. aeruginosa strains.
Different definitions have been employed to characterize multidrug resistant (MDR) isolates of P. aeruginosa in biomedical publications (24). In the majority of studies, MDR was defined as the acquired non-susceptibility to at least one agent in three or more antimicrobial categories, mainly aminoglycosides, anti-pseudomonal penicillins, cephalosporins, carbapenems, and fluoroquinolones (25)(26). Our study revealed that the frequency of MDR P. aeruginosa is 75%. Some studies carried out in Amazon region in Brazil (21), Zhenjiang in China (27) and Guilan in (23) showed that 75%, 90.1% and 42.3% of IRAN. J. MICROBIOL. Volume 10 Number 5 (October 2018) 300-306 P. aeruginosa isolates were resistant to three or more antimicrobials, respectively.
Considering that almost half of the samples were taken from a burn unit and 18.8% of the samples were collected from an ICU unit, the high prevalence of MDR cases can be justified in the present study. In this study, the highest antimicrobial resistance rate was observed for gentamicin (63.2%). The resistance rate against gentamicin was reported 55.8% in France in 2008 (28) and 95% in Thailand in 2013 (29) and in Iran, in studies carried out by Kohanteb and Imani, these rates were 68.3% and 25.5%, respectively (22,30).
On the whole, resistance to aminoglycosides in developing countries was more than that in developed countries which could be the result of the indiscriminate use of antibiotics. In our study, class 1 integron was detected in 82.6% of the isolates. Reports of P. aeruginosa clinical isolates carrying class 1 integron in Iran vary between 39.4% and 56.3% (31,23,32).
Other studies carried out on various clinical samples in Amazon region in Brazil (21), Malaysia (14), Nanjing (15) and Zhenjiang (27) in China showed that 41.5%, 63%, 38% and 40.8% of P. aeruginosa isolates carried the class 1 integron gene, respectively. Comparing the results of our study with those from other studies, the increased prevalence of integrase 1 is obvious which can be the result of geographical differences and indiscriminate use of antibiotics.
In the present study, the class 1 integron in MDR strains has higher prevalence as compared with the non-MDR strains; these results are consistent with those of Yousefi and Zhenjiang study (27,31). Our study also revealed that class 1 integron was significantly associated with resistance to given antibiotics, including aminoglycosides, quinolones, and β-lactam compounds. In view of the fact that many antibiotic resistance gene cassettes encoding resistance to a wide range of antibiotics in P. aeruginosa are carried by class 1 integron, this is not a wonder. Resistance against the antibiotics was also observed in other integron-negative isolates, however. Chromosomal-encoded enzymes or other mobile elements could account for the acquisition of the antibiotic-resistance genes of the isolates.
In this study, the intI2 gene was detected in 15.3% of the isolates. The research done by Xu (33) in China and Moazami Goudarzi (20) in Iran estimated the prevalence of class 2 integron to be 19.5% and 2.7%. In the research conducted in Thailand (34), intI2 and intI3 genes were not present. Furthermore, the intl3 gene was not observed in this research. On the other hand, identification of intI3 has been reported before in three or more Gram-negative bacteria species that had been isolated from areas with significant geographical difference. Therefore, the emergence and proliferation of integrons of class 3 carry a variety of gene cassettes that explain multiple antimicrobial resistance can be regarded as a global issue rather than a local problem (35).

CONCLUSION
In this study, we observed high prevalence of MDR P. aeruginosa (75%). In order to prevent formation of P. aeruginosa strains which may be MDR, an antimicrobial susceptibility test, especially MIC, should be performed before starting the treatment, and adequate supervision is required for the use of antibiotics. Regarding the high prevalence of intI1 gene in this study (82.6%) and its effect on increasing the P. aeruginosa antibiotics resistance, determination of positive cases and precise detection of antibiotic susceptibility pattern is strongly recommended. Apart from whether the resistance genes are present in integrons, in this study there is noticeable relationship between the presence of integrons and increased resistance to many groups of antibiotics and this could be a concern because these structures can alter involved genes in resistance between strains so that these strains become resistant to new antibiotics.